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"Synapses are physical sites of communication that transmit and transform information between neurons in a very rapid and dynamic way. Not surprisingly, malfunctioning synapses are at the root of some of our most prevalent neurological and psychiatric disorders.As synapses are smaller than what diffraction-limited light microscopy can resolve, and densely packed in light-scattering brain tissue, it has been extremely difficult to study their physiology in mechanistic terms. As a result, we still lack an understanding of the basic dynamic organization of neurotransmitter receptors and their molecular partners at mammalian synapses.While electron microscopy provided detailed snapshots of where glutamate receptors are located inside synapses, this technique does not convey dynamic or functional information. Since existing optical approaches, such as 2-photon glutamate uncaging, do not have sufficient spatial resolution, progress in this area relies on fundamental breakthroughs in live-cell-compatible techniques relying on focused visible light.We propose to utilize novel STED superresolution microscopy to image and concurrently activate synapses in live spines by superresolution STED photo-uncaging of glutamate. STED microscopy offers optical resolution an order of magnitude higher than current 2-photon or confocal techniques, and we aim to unravel functional and structural nano-dynamics of spines and synapses during plasticity. Specifically, as part of a collaborative effort, we will (1) evaluate newly engineered photosensitive glutamate-containing compounds for superresolution STED-based photo-activation, (2) advance STED microscopy technology to concurrently activate and image synapses beyond the diffraction limit, and (3) use this new methodology to probe synaptic physiology in brain slices with unprecedented resolution. These advances will enable us to address timely questions regarding the dynamic behavior of neurotransmitter receptors in individual spines."

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High-resolution real-time neuronal imaging

Neuronal cells have strange branching extensions with little knobby bulbs on them called spines, the places where one neuron communicates with another. In pioneering work, scientists have stimulated individual synapses and imaged spine changes.

Neurons have a unique morphology compared to most other cells in the body that are an approximate sphere. In addition to the cell body, they have specialised extensions for sending and receiving information. A branched dendritic tree comes off one region of the cell body and a single long axon off another.
Cells are small and dendrites even smaller. To complicate the picture a little more, the dendrites themselves have little knobby mushroom-shaped protrusions called dendritic spines. It is here that the synapses or junctions between neurons do their magic. It is also here that many neurological diseases find their origin.
Given their extremely tiny size and fast dynamics, studying them in situ has been very difficult. Scientists launched the EU-funded project 'Nanoscale photoactivation and imaging of synaptic spine dynamics' (DYNASPINE) to develop and apply the techniques to do so. Their ultimate goal was to correlate structure and function on the single-synapse level in real time.
Neuronal signalling relies on a complicated interaction of chemical and electrical components. Voltages along the membrane change, pores in the membranes open and close, and ions and molecules move in and out. Even the number, size and shape of spines demonstrate plasticity (the ability to change). Such changes can accompany increases in synaptic strength that last for long periods of time (long-term potentiation), also induced by repeated stimulation. This phenomenon is thought to be involved in learning and memory.
Scientists applied a combination of electrophysiological recordings and one of the most advanced and high-resolution microscopy techniques available, stimulated emission depletion microscopy. The team uncaged photo-releasable glutamate, an excitatory neurotransmitter, to stimulate receptors at a single synapse.
Experiments revealed the plasticity of the spine, in particular shortening and widening of the spine neck, during synaptic potentiation. They also showed that these structural changes had unexpectedly different effects on chemical and electrical signalling, pointing to a new layer of complexity in neuronal dendritic spine function.
DYNASPINE opened a new window on functioning dendritic spines. Follow-up of this exciting research direction will be met with great interest by the neuroscience community.

Executive summaryThis project aimed at increasing our understanding of dendritic spines as specialized signalling compartments that constitute tunable computational units of neurons. It relies on application of established electrophysiological techniques, as well as development and application of complex photonic microscopic techniques to visualize and functionally probe dendritic spines at high resolution.To undertake this task the fellow, Jan Tønnesen, moved from Denmark to the lab of Valentin Nägerl in Bordeaux, France, where the project was carried out. Jan Tønnesen brought electrophysiological expertise to the project, and acquired solid expertise in microscopic approaches from the host during the project. Additional new and advanced photonic techniques were implemented in the host lab by the fellow.With minor deviations from the initial milestones the project objectives were met, and novel aspects of the physiology and anatomy of dendritic spines were reported. These findings were published in a high impact peer reviewed journal within the neuroscience field. Additionally, the fellow contributed to reviews and methodological papers during the project, disseminating his knowledge and further advertising the lab and the topic.The findings brought about during the project have spurred on-going research lines in the host lab, where the fellow is currently still working.

Summary of project context and objectivesThe overall aim of this project was to push forward our neurobiological understanding of the relationship between structure and function of individual synaptic spines. Specifically, this project sought to understand the role of the spine neck as a chemical and electrical compartmentalizer of the synapse.To meet this goal we applied a combination of electrophysiological recordings and superresolution stimulated emission depletion (STED) microscopy. Unlike conventional imaging modalities, such as confocal and 2-photon microscopy techniques, STED microscopy can reliably resolve the dendritic spines and report their structural dynamics in plasticity schemes. To functionally address individual spines 2-photon uncaging of photo-releasable glutamate was applied, allowing stimulation of receptors at single synapse level. In addition, fluorescence recovery after two-photon photobleaching (FRAP) in individual spines was performed. This approach allows a detailed structurefunction analysis of live dendritic spines.Together, these experimental techniques allow a detailed functional and structural analysis of live spines.

Main resultsThe experimental data collection proceeded in four phases. First, live dendritic spines were imaged by STED microscopy, and their morphology characterized in detail. Second, spines were imaged by STED microscopy and subjected to two photon FRAP experiments, which allowed morphology to berelated to diffusional properties. Third, spine morphology was observed over time in settings of synaptic plasticity, by potentiating individual spines through two photon glutamate uncaging during STED imaging. Fourth, STED, FRAP and glutamate uncaging were combined in individual spines to provide detailed information about the relation between structural and functional plasticity of live spines.The results obtained revealed that live spines cover a broad range of sizes and shapes, and that spine shape correlates strongly with diffusional properties of the spine, thereby shaping it as a biochemical compartment. Additionally, the spine neck is a likely modulator of synaptic potentials passing the spine, as estimated from morphological and diffusional data.A major result was to visualize structural spine neck plasticity in settings of synaptic potentiation, with the neck shortening and widening. These structural changes unexpectedly had different influences on biochemical and electrical signaling, revealing a new layer of complexity in spine physiology.